Stimulation of cells with extracellular signals results in the triggering of multiple signal transduction pathways. These pathways are cascades of information transfer that are initiated at the cell membrane and culminate in the nucleus, ultimately controlling gene expression, cellular proliferation and differentiation. One of these signalling systems involves the lipid kinases phosphatidylinositol-3-OH kinase, or PI(3)-kinases, which phosphorylate the hydroxyl group at position 3 on the inositol ring of phosphoinositides, a key activity that is switched on by a huge number of extracellular stimuli.
Multiple species of 3'-phosphorylated inositol lipids are thought to be involved in a number of cellular signaling and membrane trafficking pathways, including membrane ruffling (1), chemotaxis (3), secretory responses (3), membrane trafficking of growth factor receptors (4), insulin secretion, cell regulated adhesion (5) and insulin-mediated translocation of glucose transporters to the cell surface (reviewed in Czech (1995) Annu. Rev. Nutri. 15:441-471). A relatively large, constitutive pool of PI 3-phosphate is present in resting cells, while very low levels of PI 3,4-biphosphate and PI 3,4,5-triphosphate are rapidly increased in response to a number of external cellular stimuli (reviewed in Cantley et al. (1991) Cell 64:281-302 and Kapeller, R. and L. C. Cantley (1994) Bioessays 16:565-578). The pool of PI 3-phosphate may be largely due to PI (3)-kinases such as PtdIns 3-kinase (3), a mammalian homolog of the yeast VPS34 protein (Herman, P. K. and S. D. Emir (1990) Mol. Cell. Biol. 10:6742-6754), which can utilize only PI as substrate. In contrast, a second category of PI 3-kinases, isoforms of the p110 PI 3-kinase, are capable of phosphorylating PI 4-phosphate and PI 4,5-bisphosphate at the 3' position (Hiles et al. (1992) Cell 70:419-429; Hu et al. (1993) Mol Cell. Biol. 13:7677-7688; Kippel et al. (1994) Mol. Cell. Biol. 14:2676-2685; Stoyanov et al. (1995) Science 269:690-693). These enzymes apparently contribute to the regulated pools of PI 3,4-P.sub.2 and PI-3,4,5-P.sub.5 stimulated by receptor or non-receptor tyrosine kinase activation (in the case of isoforms p110 and p110.beta.) or G protein activation (in the case of p110.gamma.). The existence of multiple PI 3-kinase isoforms suggests the influence of multiple signaling pathways on these enzymes and, possibly, divergent reactions of the individual 3'-phosphoinositides.
Relatively little is known, however, about the mechanisms that transmit the signal beyond this point. Several groups have reported a novel protein module of approximately 100 amino acids termed the pleckstrin homology (PH) domain located at the carboxy-terminal of several proteins involved in signal transduction processes (Haslam et al. (1993) Nature 363:309-310; Mayer et al. (1993) Cell 73:629-630; Musacchio et al. (1993) Trends Biochem. Sci. 18:343-348). PH domains have been implicated in the binding to membranes containing PI 4,5-bisphosphate, as well as to the binding of several proteins .beta..gamma. subunits (G .beta..gamma.) of heterotrimeric G proteins (Touhara et al. (1994) J. Biol. Chem. 269:10217-10220; Satoshi et al. (1994) Proc. Natl. Acad. Sci. USA 91:11256-11260; Lemmon et al. (1995) Proc. Natl. Acad. Sci. USA 92:10472-10476), protein kinase C (Yao et al. (2994) Proc. Natl. Acad. Sci. USA 91:9175-9179), WD motifs (Wang et al. (1994) Biochem. Biophys. Res. Commun. 203:29-35). Although the three-dimensional structures of some PH domains have been resolved and some candidate ligands have been described, the precise determinants of PH domain-binding have not been established. It is uncertain whether all PH domains share a common ligand or whether there are differences between the PH domain that confer target specificity. Moreover, it is unclear whether 3'-phosphorylated inositol lipids act as ligands of PH domains, thus mediating the multiple signal transduction pathways triggered by (PI)3-kinases.
PH domains have been found in a number of proteins including protein kinase C .alpha., phospholipase C-.delta.1, the serine/threonine kinase known variously as protein kinase B, Akt and Rac (Burgering, B. M. T. and P. J. Coffer (1995) Nature 376:599-602; Franke et al. (1995) Cell 81:727-736; Coffer, P. J. and J. R. Woodgett (1991) Eur. J. Biochem. 201:475-481) among others. Recently, cDNAs encoding a modular protein containing a domain homologous to the yeast SEC7 gene product and a PH domain have been reported (Liu, L. and B. Pohajdak (1992) Biochim. Biophys. Acta 1132:75-78; Kolanus et al. (1996) Cell 86:233-242). Full-length and SEC7 domain forms of these recently cloned molecules have been shown to induce b-.beta. integrin-dependent binding of lymphoid cells to extracellular molecules such as ICAM-1 (Kolanus et al. (1996) supra). This finding implicates PH/SEC7 containing molecules not only in transducing extracellular signals into a cell, but in conveying information from the cell interior to the exterior via a mechanism termed "inside out signaling". In the immune system, inside out signaling has been associated with the orderly attachment of cells to their surrounding matrix, the adhesion of platelets to fibrinogen, the coupling of lymphocytes to their antigen presenting cells, and the phagocytosis of complement-opsonized targets by myelomonocytic phagocytes (reviewed by Diamond, M. S. and T. A. Springer (1993) J. Cell. Biol. 120:54556; Hynes (1992) Cell 69:11-25; and Sastry, S. K. and A. F. Horwitz (1993) Curr. Opn. Cell. Biol. 5:819-831).
Furthermore, SEC7 domains have also been implicated in glycoprotein secretion. SEC7 was originally identified in yeast as one of the at least 23 genes implicated in the process of intercompartmental protein transport (Novick, P. et al. (1981) Cell 25, 461-69; Novick, P. et al. (1980) Cell 21:205-157). Most of the SEC mutations block transport of proteins from the endoplasmic reticulum to the Golgi apparatus or from mature secretory vesicles to the plasma membrane (Esmon, B. et al. (1981) Cell 25:451-60; Stevens, T. et al. (1982) Cell 30: 439-48; Riezman, H. (1985) Cell 40: 1001-09). Mutations that define the sec7 locus exert a unique and dramatic effect on traffic of secretory, plasma membrane, vacuolar, and endocytic marker molecules (Novick et al. (1980) supra; Esmon et al. (1981) supra). At the restrictive growth temperature, sec7 mutant cells accumulate secretory glycoproteins within the Golgi apparatus, leading to the exaggeration of Golgi cisternae. The SEC7 gene has been cloned and shown to encode a large protein containing 2008 amino acids (Achstetter et al. (1988) J. Biol. Chem. 263(4):11711-11717).
There is evidence which suggests that compromise of PH domain function of Bruton's tyrosine kinase underlies the dysregulation of B cell ontogeny that is associated with the murine X-linked immunodeficiency syndrome (Rawlings et al. (1993) Science 261:358-361). Thus, identifying novel molecules that mediate some of these signaling events is critical in the understanding of these biological processes and in the development of therapeutic methods.